Proper functioning of the central nervous system (CNS) is critically dependent on the formation of insulating myelin sheaths around axons. Myelination is a complex multi-step process that occurs primarily during early postnatal life, but it is also re-initiated in the adult CNS in response to acute demyelination insults. Dysfunction o myelin-forming cells and/or loss of myelin sheath underlie many neurological disorders including multiple sclerosis (MS) and psychological disorders such as schizophrenia. Promoting myelin repair and achieving neuroprotection has attracted increasing attention, and several pharmaceutical companies and research laboratories including ours are actively pursuing the discovery of drug-like small molecules that promote such processes. However, one of the major roadblocks in this effort is the lack of robust in vitro CNS myelination models. Our multidisciplinary research team has recently developed a microfluidic CNS neural stem/progenitor cell (NSPC) aggregate culture system that for the first time demonstrated robust myelin ensheathment of cortical neurons in vitro using a microdevice. The major advantages of this aggregate culture system are that it contains all the essential cellular components of an in vivo environment. However the use of this microdevice is labor intensive and throughput is quite limited. Here we propose to develop a high-throughput microfluidic NSPC aggregate culture system that provides at least two orders of magnitude higher throughput than existing approaches including ours, establishing for the first time a high-throughput in vitro CNS myelination model that has physiologically relevant neuronal responses and is amenable to drug screening applications. The innovative three-dimensional high-throughput screening (3D-HTS) microsystem will have at least three sets of 100 independently controlled microscale culture compartments (300 total), each compartment having eight aggregate trapping sites. The fully automated system will enable screening the effects of 20 candidate drug molecules at 5 different concentrations each, with 3 repeats per condition, all in a single experimental run. The microfluidic system configuration is flexible, where it can be easily re-configured to screen large number of candidate drug molecules with fewer number of concentrations tested. As image processing is another major bottleneck for high-throughput screening, we will develop an image processing algorithm that will minimize the number of images required for myelin segment quantification and fully automate the image processing steps for the large number of immunostained images that will be generated. We will conduct a proof-of-concept drug screening assay using this microfluidic system. This will be the first in vitro myelinating system recapitulating cortical axon-glial interactions and one that is fully adaptable to automation of th entire drug screening assay. We expect that the successful development of this high-throughput platform will lead to a routine drug screening system for identifying potential targets or candidate drug molecules that can stimulate myelin repair and help recover function in demyelinating diseases and other neurological disorders.